Process for fermentative side chain cleavage of 20-keto pregnanes

ABSTRACT

A NOVEL METHOD FOR THE INHIBITION OF OVER-OXIDATION DURING THE FERMENTATIVE SIDE CHAIN CLEAVAGE OF 20-KETO COMPOUNDS OF THE PREGNANE SERIES TO OBTAIN THE CORRESPONDING 17-KETOANDROSTANES. THE IMPROVED PROCESS OF THIS INVENTION PROVIDES A METHOD WHEREBY 20-KETOPREGNANES WHICH ARE UNSUBSTITUTED AT THE 1M AND 21-POSITIONS ARE BIOCONVERTED TO THE CORRESPONDING 17-KETO COMPOUNDS OF THE ANDROSTANE SERIES (17-KETOANDROSTANES) IN HIGH YIELD BY EMPLOYING HIGH SUBSTRATE LEVELS TO INHIBIT THE FORMATION OF D-RING LACTONES. THE 17-KETOANDROSTANES PRODUCED BY THE IMPROVED PROCESS OF THIS INVENTION ARE KNOWN USEFUL THERAPEUTIC AGENTS AND THEY ARE ALSO USEFUL AS INTERMEDIATES WHICH CAN BE CONVERTED BY KNOWN METHODS TO KNOWN USEFUL THERAPEUTIC AGENTS.

United States Patent 3,556,944 PROCESS FOR FERMENTATIVE SIDE CHAIN CLEAVAGE OF 20-KETO PREGNANES Thomas L. Miller, Portage, Mich., assignor to The Upjohn Company, Kalamazoo, Mich., a corporation of Delaware No Drawing. Filed Jan. 10, 1969, Ser. No. 790,440 Int. Cl. C07c 167/18 U.S. Cl. 195-51 9 Claims ABSTRACT OF THE DISCLOSURE A novel method for the inhibition of over-oxidation during the fermentative side chain cleavage of 20-keto compounds of the pregnane series to obtain the corresponding 17-ketoandrostanes. The improved process of this invention provides a method whereby 20-ket0pregnames which are unsubstituted at the 17 and 21-positions are bioconverted to the corresponding 17-keto compounds of the androstane series (l7-ketoandrostanes) in high yield by employing high substrate levels to inhibit the formation of D-ring lactones. The 17-ketoandrostanes produced by the improved process of this invention are known useful therapeutic agents and they are also useful as intermediates which can be converted by known methods to known useful therapeutic agents.

BACKGROUND OF THE INVENTION The fermentative cleavage of the 17-side chain of pregnanes is well known in the art. Progesterone, for example, is bioconverted to 4-androsten-3,17-dione and 1,4-androstadiene-3,l7-dione. It is also well known that under conventional fermentation conditions wherein low substrate levels are employed, i.e., 2 g. per liter and less, side chain cleavage is complicated by overoxidation of the desired 17-ketoandrostane to give the corresponding D-ring lactone. U.S. Pats. 2,762,747, 2,866,737, 2,721,828 and 2,902,410 show the low yields of 17-keto steroids obtained when low substrate levels are employed. These low yields are owing to over-oxidation to the corresponding D-ring lactones as shown in U.S. Pat. 2,902,410.

Heretofore, inhibition of D-ring lactone formation in microbial side chain cleavage of 20-ketopregnanes has been achieved only by the addition of extraneous chemical inhibitors, such as allosteroids, metal binding reagents, and compounds which may be considered as fragments of the steroid molecule, e.g., naphthalene, wnaphthol, phenanthrene, quinoline and the like. For example, see Wix et al., Biotech. Bioeng. 1, 239 (1959) and British Pats. 915,107 and 918,960.

When low substrate levels are employed in the absence of chemical inhibitors reasonably good yields (SO-60%) can only be obtained by stopping the fermentation at the precise time that an optimum yield of the desired 17-keto steroid is obtained since continued fermentation results in over-oxidation to the D-ring lactone. This method is undesirable because it requires continuous sampling and assay during the entire fermentation period. This method is also unreliable because at low substrate levels D-ring lactone formation often occurs during the entire fermentation.

BRIEF SUMMARY OF THE INVENTION It has now been discovered that over-oxidation of the desired 17-ketoandrostane product, produced by fermentative cleavage of 20-ketopregnanes, can be inhibited by utilizing a high substrate level, i.e., greater than 4 g. per liter, of the starting 20-ketopregnane. D-ring lactone formation is thus effectively inhibited Without employing extraneous chemical inhibitors.

The improved process of this invention is of considerable economic importance. Its principle advantages over the prior art are (1) high yields and greatly increased product per batch, resulting in substantially reduced labor and equipment requirements; (2) lower cost per pound of product; (3) extraneous chemical inhibitors are eliminated resulting in ease of recovery and less contamination of the desired product; (4) undesirable D-ring lactone formation is substantially eliminated.

DETAILED DESCRIPTION OF THE INVENTION The improved process of the present invention comprises subjecting a 20ketopregnane to the oxygenating activity of a microorganism capable of l7-side chain cleavage to obtain the corresponding l7-ketoandrostanc.

The improved process of the present invention provides a means for obtaining a variety of known useful products such as those described in the U.S. patents cited hereinabove. For example, progesterone is converted to 4-androstene-3-l7-dione, a known androgenic agent which can be converted by known methods to 17-ethynyltestosterone.

Starting compounds for the improved process of the present invention are 20-ketopregnanes which are unsubstituted at the 17 and 21-positions; they can also possess keto, hydroxy, alkyl, halo, cyclopropyl and other groups attached to the steroid rings, especially at positions 3, 4, 6, 7, 8, 11, 12, 14 and 16 and they can have double bonds present especially in positions 1(2), 4(5), 5(6), 6(7), 9(11) and 16(17). Representative starting materials include, for example, progesterone, 11aand ILB-hydroxyprogesterone, ll-ketoprogesterone, 6-dehydroprogesterone, 6-dehydro-6a-methylprogesterone, 6a-methy1progesterone, 16aand 16B-methylprogesterone, 30cand 3,8- pregnane-ZO-one, 3aand 3fl-hydroxy-5-pregnen-20-one, 3ccand 3B hydroxypregnane-l1,20-dione, 3u,l1u-hydroxypregnan 20 one, 30,11[3-hydroXypregnan-20-one, 313,11fl-hydroxypregnan-ZO-one, allopregnan 3,20 dione and the like.

In carrying out the improved process of this invention, a 20-ketopregnane is subjected to the oxygenating activity of a microorganism capable of steroidal side chain c1eavage. Representative microorganisms include, for example, certain species of the genera Penicillium, Fusarium, Septomyxa, Gliocladium, Cephalosporium, Cylindrocarpon, Cylindrocephalum and the like.

Typical species useful in the improved process of this invention are:

Penicillium lilacinum, NRRL 3422 Penicillium lilacinum, ATCC 10, 114 Penicillium notatum, NRRL 832 Penicillium canescens, ATCC 1C, 419 Penicillium charlqsii, ATCC 8730 Penicillium brevi-compactum, ATCC 9056 Penicillium lividum, ATCC 10, 102 Penicilliufn nigricans, ATCC 10, 115 Penicillium javanicum, ATCC 9099 Penicillium expansum, ATCC 7861 Penicillium frequentans, ATCC 10, 444 Penicillium thomii, ATCC 10, 506 Penicillium novae-zeelandiae, ATCC 10, 473 Penicillium citrinum, ATCC 10, 105 Penicillium fuscum ATCC 10, 477 Septomyxa afiinis, ATCC 6737 Septomyxa affinis, ATCC 13, 414 Septomyxa carni, ATCC 13, 416

F usarium solani, NRRL 3421 Fusarium avenaceum, ATCC 8150 F usarium monoilifo'rme, ATCC 10, 052

F usarium vasenfectum ATCC 7808 Fusarium' javanicum var. ensiforme, QM 524 F usarium javanicum var. radicicola, ATCC 7595 Aspergillus chevalieri, CBS, ATCC 9908 Aspergillus fischeri, CBS, ATCC 1020 Aspergillus flavipes, CBS, ATCC l l, 013 Aspergillus flavus, CBS, NRRL 484 Aspergillus oryzae, CBS, ATCC 1011 Aspergillus pe'nicilloides, CBS, 'CZAA Aspergillus ruber, CBS, CZAA Aspergillus sclerotiorwm, CBS, CZAA ATCC 16, 892 Aspergillus terreus, CBS, CZAA Aspergillus terricola, CBS, CZAA Cephalosporium suberticillatum, PIRI Cylindrocarpon radicicola, CBS, ATCC, 11, 011 Cylindrocephalum aureum, ATCC 12, 720 Gliocladium catenulatum, ATCC 10, 523 Gliocladium roseum, ATCC 10, 521 Gliocladium deliquescens, CBS

Gliocladium luteolum, CBS, ATCC 10,097 Glz'ocladium vermoeseni, ATCC 10,522

and the like, which are available from known public sources such as the Northern Utilization Research and Development Branch, U.S. Department of Agriculture, Peoria, Ill. (NLRR); the American Type Culture Collection (ATCC), Washington, D.=C.; "Centraalbureau voor Schimmelcultures (CBS), Baarn, Holland; Quartermaster Culture Collection (QM), Quartermaster Research and Engineering Command, United States Army, Natick, Mass; Research Institute for the Pharmaceutical Industry (PIRI), Budapest, Hungary; and Czechoslovak Academy of Agricultural Sciences (CZAA), Prague, Czechoslovakia.

In the practice of this invention, the species Penicillium lilacinum, Fusarium solani and Septomyxa afiinis are especially advantageous.

Some of the microorganisms useful in the process of this invention concommitantly l-dehydrogenate the steroid A-ring during side chain cleavage, for example, those of the genera, Fusarium, Septomyxa, Cyclindrocarpon, Cylindrocephalum and Gliocladium.

The operational conditions and reaction procedures for the bioconversion process of this invention are advantageously those known in the art of bioconversion as illustrated in Murray et al., U.S. Pats. 2,762,747 and 2,721,828.

In the practice of this invention, the bioconversion can be effected by a growing or resting culture of the microorganism or by spores, washed cells or enzymes of the microorganism.

Culture of the microorganism for the purpose and practice of this invention is in or on a medium favorable to its development. Sources of nitrogen and carbon should be present in the culture medium and an adequate sterile air supply should be maintained during the conversion, for example, by the conventional techniques of exposing a large surface of the medium or by passing air through a submerged culture.

Nitrogen in assimilable form can be provided by sources normally employed in such processes, such as cornsteep liquor, cottonseed meal, soybean meal, yeast extracts, peptone, soluble or insoluble vegetable or animal protein, lactalbumein, casein, whey, distillers solubles, amino acids, nitrates and ammonium compounds, such as ammonium tartrate, nitrate, sulfate and the like.

Available carbon can also be provided by sources normally used in bioconversions such as carbohydrates, e.g., glucose, fructose, sucrose, lactose, maltose, dextrins, starches, meat extracts, peptones, amino acids, proteins, fatty acids glycerol, whey and the like. These materials may be used either in a purified state or as concentrates such as whey concentrate, cornsteep liquor, grain mashes, cottonseed meal, and the like, or as mixtures of the above. Many of the above sources of carbon can also serve as a source of nitrogen.

The medium can desirably have a pH before inoculation of between about pH 4 to about 8 through a higher or lower pH can be used. A temperature between about 25 to 32 C. is preferred for growth of the microorganism but higher or lower temperatures within a relatively wide range are suitable.

In the practice of this invention is important that the selected substrate be added in comminuter form to eliminate the harmful effects of high concentrations of steroid solvents on the fermentation. Micronized substrate material wherein substantially all particles are smaller than 20 microns is preferred and a particle-size distribution of (by weight) smaller than 10 microns is especially advantageous in obtaining optimum yields and rates of production of the desired 20-keto steroids. The substrate can be added to the culture as a dry powder or as an aqueous suspension, either as a single feed or by gradually adding the substrate slowly throughout the conversion period. For mechanical reasons, the substrate is preferably added as an aqueous suspension. In preparing the aqueous suspension the use of dispersing agents or suspending agents is advantageous. Examples of such agents are commercially available Spans (hexitol anhydride esters of long-chain fatty acids), Tweens (polyoxyalkylene ethers of hexitol anhydride long-chain fatty acid esters), Nacconols (alkyl aryl sulfonates), Ultrawets (alkyl benzene sodium sulfates), and the like. The preferred, but not limiting, range of concentration of the substrate in the culture medium is from 4 to about 10 g. per liter.

The temperature during the fermentation can be the same as that found suitable for growth of the microorganism. It need be maintained only within such range as supports life, active growth or the enzyme activity of the microorganism. A range of 20 to 35 C. is preferred. A pH of about 6 to 8 is generally preferred during the bioconversion. Aeration can be effected by surface culture or preferably by use of submerged fermentation conditions with air sparging, in accordance with methods well known in the art. The time required for oxygenation by the enzymatic system of the microorganism employed can vary considerably. The range of about 2 to hours is practical but not limiting; 24-72 hours is generally satisfactory. The progress of the bioconversion and its completion are conveniently determined by paper-strip chromatography, or thin-film chromatography [Heftman, Chromatography (1961) Reinhold Publishing Co., New York, N.Y.].

Alternatively, oxygenation of the selected substrate can be effected by subjecting it to the activity of enzymes prepared from the microorganism, to the action of spores of the microorganism, and to the action of isolated cells of the microorganism. Isolated enzyme preparations can be prepared in accordance with the general procedure disclosed by Zuidweg et al., Biochim. Biopy. Acta 58, 131133 (1962). The bioconversion can be effected with spores in accordance with the general process disclosed in U.S. Pats. 3,031,379 and 3,031,382. The separation of Washed cells from the fermentation medium is well known in the art, see for example, U.S. Pat. 2,831,789.

The term oxygenation as used throughout this specification means the enzymatic action of a growing or rest ing culture of the microorganism which effects side chain cleavage and the introduction of a 17-keto group into the substrate.

After completion of the steroid fermentation, the resulting 17-keto steroid is recovered from the fermentation reaction mixture by conventional methods. An especially advantageous manner of recovering the product involves extracting the fermentation reaction mixture, including the fermentation liquor and rnycelia, with a water-immiscible organic solvent for steroids, for example, methylene chloride, chloroform, carbon tetrachloride, ethylene chloride, trichloroethylene, ether, amyl acetate, benzene, toluene, and the like. Alternatively, the fermentation liquor and mycelia can be first separated by conventional methods, e.g., filtration or centrifugation, and then separately extracted with suitable solvents.

The mycelia can be extracted with either water-miscible or water-immiscible solvents. The fermentation liquor, freed of mycelia, can be extracted with water-immiscible solvents. The extracts can be combined, the solvent removed and the purified 17-keto steroid is obtained by recrystallization of the residue from organic solvents.

The following examples are intended to illustrate the process of this invention. The examples are for the purpose of illustrating the best mode contemplated of carrying out the invention and to supplement the foregoing disclosure with additional descriptions of the manner and process of carrying out the invention so as to further enable workers skilled in the art to do so, but they are not to be construed as limiting.

EXAMPLE 1 Four 500 ml. shake flasks each containing 100 ml. of a medium consisting of 1 g. of dextrose (Cerelose) and 2 g. of cornsteep liquor made up to 100 ml. with tap water, are adjusted to pH 5.5, sterilized at 121 C. for 15 minutes, cooled to about 28 C., inoculated with a 24- hour vegetative growth of Fusarium solani, NRRL 3421, and incubated for approximately 24 hours on a rotary shaker at 28 C. Micronized progesterone (particlesize distribution 90% smaller than microns by weight) is then added to each as a dry powder in the amounts of 1, 2, 4 and 8 grams per liter as shown in the table, below. Thefermentation is continued at 28 C. and samples are taken from each of the flasks at regular intervals and assayed for androsta-1,4-diene-3,17 -"dione and n -testololactone by vapor-phase chromatography (VPC). The optimum fermentation time for each and the yields in percent of theory of androsta-1,4-diene-3,17- dione (ADD) and n -testololactone (A -test.) are shown A 500 ml. shake flask containing 100 ml. of an aqueous medium consisting of 1 g. of dextrose (Cerelose), 0.5 g. of Torula yeast, 0.5 g. of ground extracted soybean (Kaysoy 200 C, Archer Daniels Midland Co., Minneapolis, Minn.) and 0.3 g. of cornsteep liquor is adjusted to pH 5.5, sterilized for minutes at 121 C., cooled to about 28 C., inoculated with a 24-hour vegetative growth of Penicillium lilacinum, NRRL 3422, and incubated for about 24 hours on a rotary shaker at 28 C. Micronized progesterone 0.6 g. 6 g./ 1.) (90% smaller than 10 microns) is then added as a dry powder and the fermentation is continued. Samples are taken at regular intervals during the fermentation period and assayed by vapor-phase chromatography. After 48 hours of fermentation time, VPC analysis shows a 81.0% yield, 0.44 g. of 4-anrostene-3,l7-dione.

EXAMPLE 3 EXAMPLE 4 The bioconversion and assay procedures of Example 2 are repeated using Fusarium solani, NRRL 3421, and

1.0 g. (10 g./l.) of progesterone (90% smaller than 10 microns). The fermentation is continued for 96 hours following addition of the substrate to give 91.7% yield, 0.83 g. of androsta-l,4-diene-3,17-dione.

EXAMPLE 5 A 500 ml. shake flask containing 100 ml. of an aqueous medium consisting of 1 g. of dextrose and 2 g. of cornsteep liquor made up to 100 ml. with tap water is adjusted to pH 5.5, sterilized at 121 C. for 15 minutes, cooled to about 28 C., inoculated with a 24-hour growth of Sepomyxa Affinis, ATCC 6737, and incubated on a rotary shaker at 28 C. for 24 hours. Micronized progesterone, .05 g. (0.5 g./l.) smaller than 20 microns by weight) is then added as a dry powder and the fermentation is continued. Samples are taken at regular intervals during the fermentation period and assayed by vaporphase chromatography. After 24 hours of fermentation time, VPC analysis shows a yield of 30.9%, 0.014 g. of androsta-1,4 diene-3,17 dione. After an additional 24 hours of fermentation time substantially all of the androsta l,4-diene-3,17-dione is converted to A -testololactone.

EXAMPLE 6 The bioconversion and assay procedures of Example 5 are repeated using Septomyxa afifnis, ATCC 6737, and 0.1 g. (1 g./l.) of progesterone (90% smaller than 10 microns). Samples taken at 24-hour intervals during the fermentation show the following yields of androsta-1,4- diene-3,17-dione 24 hours-57.7% (0.052 g.) 48 hours-38.0% (0.034 g.) 72 hours-32.5% (0.029 g.)

EXAMPLE 7 The bioconversion and assay procedures of Example 5, are repeated using Septomyxa ayfinis, ATCC 6737, 0.4 g. (4 g./l.) of progesterone (90% smaller than 10 microns by weight). The fermentation was continued for 72 hours following addition of the substrate. VPC analysis shows a 95.3% yield, 0.345 g. of androsta-1,4-diene-3,17-dione.

EXAMPLE 8 The bioconversion and assay procedures of Example 5 are repeated using Septomyxa afiinis, ATCC 6737, and 0.6 g. (6 g./l.) of progesterone (90% smaller than 10 microns by weight). The fermentation is continued for 72 hours following addition of the substrate. VPC analysis shows a 96% yield, 0.52 g. of androsta-1,4-diene-3,17- dione.

EXAMPLE 9 The bioconversion and assay procedures of Example 5 are repeated using Septomy-xa affinis, ATCC 6737, and 0.4 g. (4 g./l.) of llet-hydroxyprogesterone (90% smaller than 10 microns by weight). The fermentation was continued for 48 hours following addition of the substrate. VPC analysis shows a 77% yield, 0.284 g. of llu-hydroxyandrosta-1,4-diene-3,l7-dione.

EXAMPLE 10 A 500 ml. shake flask containing 100 ml. of an aqueous medium consisting of 1.5 g. of dextrose, 0.2 g. of KHPO 0.05 g. of MgSO -7H O', and 0.3 g. of Peptone (partially hydrolyzed protein) made up to 100 ml. with tap water adjusted to pH 5.5, sterilized at 121 C. for 15 minutes, cooled to about 28 C., inoculated with a 24-hour growth of Septomyxa aflinis, ATCC 6737, and incubated on a. rotary shaker at 28 C. for 24 hours. Micronized progesterone 0.4 g. (4 g./l.) (90% smaller than 20 microns by weight) is then added as a dry powder and the fermentation is continued for 72 hours. Vapor-phase chromatography analysis shows a yield of 87.4%, 0.316 g. of androsta-1,4-diene-3,l7-dione.

7 EXAMPLE 11 The bioconversion and assay procedures of Example 9 are repeated using Sepromyxa afiinis and 0.8 g. (8 g./l.) of progesterone (90% smaller than 10 microns by weight). The fermentation is continued for 72 hours following addition of the substrate. VPC analysis shows a yield of 90.4%, 0.655 g. of androsta-1,4-diene-3,17-dione.

EXAMPLE 12 A medium is prepared consisting of 5 kg. of cornsteep liquor, 2.5 kg. of dextrose (Cerelose) and 250 liters of tap water. The pH is adjusted to 5.5 and a minimum quantity of soybean oil is added to prevent foaming. The medium is sterilized at 122 C. for 15 minutes, cooled to about 28 C. and inoculated with 15 liters of a 24-hour vegetative growth of Septomyxa aflinis, ATCC 6737. The medium is then agitated at 280 r.p.m., aerated with sterile air at the rate of liters per minute and allowed to grow for 24 hours at about 28 C. Progesterone 1 kg. (4 g./l.) (90% smaller than 10 microns by weight) is then added, as an aqueous slurry and the fermentation is continued for an additional 46-hour period. Samples are taken at regular intervals during the fermentation period and assayed by vapor-phase chromatography analysis in order to follow the progress of the fermentation. At the end of the fermentation period, VPC analysis shows a yield of 1.88 kg., 83% of androsta-l,4-diene-3,l7-dione.

The product thus obtained is recovered from the fermentation medium by adding methylene chloride in an amount equal to approximately one-third the total volume of the fermentation medium, filtering to remove the mycelium, separating and washing the methylene chloride layer with water. The solvent is then removed by distillation and the residue thus obtained is recrystallized to give 0.6 kg, 66% yield of essentially pure androsta-1,4-diene- 3,17-dione; and additional 5 to 6% of androsta-l,4-diene- 3,17-dione is recoverable from the mother liquors.

EXAMPLE 13 The procedure of Example 12 is repeated using 1.5 kg. (6 'g./l.) of progesterone to give a 77% yield of androsta- 1,4-diene-3,17-dione.

Other microorganisms capable of steroidal l7-side chain cleavage, such as those listed hereinabove, can be substituted in place of Fusarium solani, NRRL 342.1, Penicillz'um lilacinum, NRRL 3422 or Septomyxa afiinis, ATCC 6737, in the foregoing examples to give comparable high yields of the desired 17-keto compounds.

In the same manner following the procedures of the foregoing examples, other compounds of the preguane series such as those listed hereinabove, can be substituted in place of progesterone to give substantially equivalent high yields of the corresponding 17-ketoandrostanes.

I claim:

1. In the process for the fermentative side chain cleavage of a 20-ket0pregnane under submerged aerobic fermentation conditions in an aqueous nutrient medium to obtain the corresponding 17-ketoandrostane, the improvement which comprises employing the ZO-ketopregnane 8 substrate in comminuted form having a particle size distribution smaller than 20 microns at a substrate level greater than 4 grams per liter of the aqueous nutrient medium. 7

2. The process of claim 1 wherein the comminuted substrate has a particle size distribution 90% smaller than 10 microns and the substrate level is within the range of 4 to 10 grams per liter of the nutrient medium.

3. The process of claim 1 wherein the ZO-ketopregnane is progesterone.

4. A method for the inhibition of the over-oxidation of a l7-ketoandrostane produced by the fermentative side chain cleavage of a ZO-ketopregnane under submerged aerobic fermentation conditions, which comprises carrying out the fermentation with a comminuted 20-ketopregnane having a particle size distribution 90% smaller than 20 microns, at a substrate level within the range of from 4 to 10 grams per liter of aqueous nutrient medium.

5. The process of claim 4 wherein the comminuted 20- ketopregnane has a particle size distribution 90% smaller than 10 microns.

6. The process of claim 4 wherein the ZO-ketopregnane is progesterone.

7. A method for the inhibition of the over-oxidation of 4-androstane-3,17-dione produced by the fermentative 17-side chain cleavage of progesterone in an aqueous 'nutrient medium under submerged aerobic fermentation conditions with the microorganism Penicillium Iilacinum, NRRL 3422, which comprises carrying out the fermentation with micronized progesterone having a particle size distribution 90% smaller than 10 microns at a substrate level within the range of from 4 to 10 grams per liter of aqueous nutrient medium.

8. A method for the inhibition of the over-oxidation of androsta-l,4-diene-3,17-dione produced by the fermentative l7-side chain cleavage of progesterone in an aqueous nutrient medium under submerged aerobic fermentation conditions with the microorganism Fusarium solani, NRRL 3421, which comprises carrying out the fermentation with micronized progesterone having a particle size distribution 90% smaller than 10 microns at a substrate level within the range of from 4 to 10 grams per liter of aqueous nutrient medium.

9. A method for the inhibition of the over-oxidation of androsta-l,4-diene-3,l7-dione produced by the fermenta tive 17-side chain cleavage of progesterone in an aqueous nutrient medium under submerged aerobic fermentation conditions with the microorganism Septomyxa ajjinz's, AT CC 6737, which comprises carrying out the fermentation with micronized progesterone having a particle size distribution 90% smaller than 10 microns at a sub strate level within the range of from 4 to 10 grams per liter of aqueous nutrient medium.

References Cited UNITED STATES PATENTS 2,721,828 10/1955 Murray et al. l5l(A417) ALVIN E. TANENHOLTZ, Primary Examiner 

